Summaryoskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

Summary piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, while the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 co-localizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts co-localization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.

Summary The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed “comparative RIC” and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

The metaphase-anaphase transition is orchestrated through proteolysis of numerous proteins by an ubiquitin protein ligase called the Anaphase-promoting complex or cyclosome (APC/C) 1 . A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves cohesin, a ring-shaped complex that entraps sister DNAs 2, 3 . It is a matter of debate whether cohesin-independent forces also contribute to sister chromatid cohesion [4][5][6] . Using 4D-live-cell imaging of Drosophila syncytial embryos blocked in metaphase (via APC/C inhibition) we show that artificial cohesin cleavage 7 is sufficient to trigger chromosome disjunction. This is nevertheless insufficient for correct chromosome segregation. Kinetochore-microtubule attachments are rapidly destabilized by the loss of tension caused by cohesin cleavage in the presence of high Cyclin-dependent kinase 1 (Cdk1) activity, as occurs when the APC/C cannot destroy mitotic cyclins. Metaphase chromosomes undergo a bona-fide anaphase when cohesin cleavage is combined with Cdk1 inhibition. We conclude that only two key events, opening of cohesin rings and down regulation of Cdk1, are sufficient to drive proper segregation of chromosomes in anaphase.Correct attachment of sister DNAs to microtubules during mitosis depends on a force that actively holds them together. It is a matter of ongoing debate whether cohesin-independent forces (be they sister DNA concatenation [8][9][10][11] or proteinaceous linkages provided by ORC 12 or condensin 13 ) contribute to sister chromatid cohesion in metaphase cells [4][5][6] . If cohesin alone resists spindle forces prior to anaphase, then artificial opening the cohesin ring by an exogenous protease should trigger sister chromatid disjunction in the absence of separase activity. If, on the other hand, cohesin were just one of several mechanisms holding sisters together, then cleavage should not suffice. This key experiment has so far only been performed in yeast, where cleavage of cohesin's α-kleisin subunit by the Tobacco Etch virus protease (TEV) in cells arrested in metaphase initiates disjunction of most sister DNAs but not those of repetitive rDNAs within the nucleolus 14,15 . It is nevertheless argued that the larger chromosomes of animal and plant cells, or indeed other fungi, have very different properties. To address this, we used strains of the fruit fly D. melanogaster whose α-kleisin subunit Rad21 has been replaced by a version containing TEV cleavage sites (Rad21 TEV ) 7 . TEV protease injection causes catastrophic mitotic failure in syncytial embryos using Rad21 TEV ( Supplementary Information, Fig. S1). When injected before or during early stages of mitosis, it causes sister chromatids to disjoin as soon as the nuclear envelope breaks down. This precocious loss of sister chromatid cohesion is accompanied by aCorrespondence and requests for materials should be addressed to K.N. (kim.nasmyth@bioch.ox.ac.uk prometaphase delay...

RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox interactions will yield important insights into RNA regulation in cellular homeostasis and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0132-3) contains supplementary material, which is available to authorized users.

Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. We have elucidated the mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo by directly visualizing intermediates along the entire path of transcript movement. After release from their site of transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization of endogenous transcripts and apical movement of injected RNA particles. We propose that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization.